ORCID Profile
0000-0001-5023-2668
Current Organisation
University of Adelaide
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Publisher: Oxford University Press (OUP)
Date: 04-2016
DOI: 10.1095/BIOLREPROD.115.137240
Abstract: Traditionally, research in the field of trace element biology and human and animal health has largely depended on epidemiological methods to demonstrate involvement in biological processes. These studies were typically followed by trace element supplementation trials or attempts at identification of the biochemical pathways involved. With the discovery of biological molecules that contain the trace elements, such as matrix metalloproteinases containing zinc (Zn), cytochrome P450 enzymes containing iron (Fe), and selenoproteins containing selenium (Se), much of the current research focuses on these molecules, and, hence, only indirectly on trace elements themselves. This review focuses largely on two synchrotron-based x-ray techniques: X-ray absorption spectroscopy and x-ray fluorescence imaging that can be used to identify the in situ speciation and distribution of trace elements in tissues, using our recent studies of bovine ovaries, where the distribution of Fe, Se, Zn, and bromine were determined. It also discusses the value of other techniques, such as inductively coupled plasma mass spectrometry, used to garner information about the concentrations and elemental state of the trace elements. These applications to measure trace elemental distributions in bovine ovaries at high resolutions provide new insights into possible roles for trace elements in the ovary.
Publisher: Public Library of Science (PLoS)
Date: 07-02-2013
Publisher: Bioscientifica
Date: 21-01-2023
DOI: 10.1530/RAF-22-0068
Abstract: Polycystic ovary syndrome (PCOS) is an endocrine metabolic disorder that appears to have a genetic predisposition and a fetal origin. The fetal ovary has two major somatic cell types shown previously to be of different cellular origins and different morphologies and to differentially express 15 genes. In this study, we isolated the somatic gonadal ridge epithelial-like (GREL) cells ( n = 7) and ovarian fetal fibroblasts ( n = 6) by clonal expansion. Using qRT-PCR, we compared the gene expression levels of PCOS candidate genes with previous data on the expression levels in whole fetal ovaries across gestation. We also compared these levels with those in bovine adult ovarian cells including fibroblasts ( n = 4), granulosa cells ( n = 5) and surface epithelial cells ( n = 5). Adult cell types exhibited clear differences in the expression of most genes. In fetal ovarian cells, DENND1A and ERBB3 had significantly higher expression in GREL cells. HMGA2 and TGFB1I1 tended to have higher expression in fetal fibroblasts than GREL cells. The other 19 genes did not exhibit differences between GREL cells and fetal fibroblasts and FBN3 , FSHB , LHCGR , FSHR and ZBTB16 were very lowly expressed in GREL cells and fibroblasts. The culture of fetal fibroblasts in EGF-containing medium resulted in lower expression of NEIL2 but higher expression of MAPRE1 compared to culture in the absence of EGF. Thus, the two fetal ovarian somatic cell types mostly lacked differential expression of PCOS candidate genes. Polycystic ovary syndrome (PCOS) is one of the most common reproductive problems. The cause is not known so there are no specific treatments or prevention strategies. We know it can be linked to issues that occur in the womb and that some people may be more likely to get PCOS due to their genetic makeup. Our recent studies showed that many of the genes linked to PCOS were found to be switched on in the fetal ovary and are likely to be involved in the development of the fetal ovary. In order to improve our understanding of PCOS, we need to identify the type of cells in the fetal ovary where these genes are switched on. In this study, we examined the PCOS genes in two types of cells that mature as the fetal ovary develops and found very little difference between them but bigger differences to their mature adult counterparts.
Publisher: Public Library of Science (PLoS)
Date: 16-03-2015
Publisher: S. Karger AG
Date: 11-12-2006
DOI: 10.1159/000097892
Abstract: i Background: /i Controversial reports on prolactin receptors (PRL-R), the long and short form, on endothelial cells (EC) may be explained by the choice of EC derived from the micro- and macrovascular bed of either endocrine and non-endocrine organs. i Methods: /i We studied here PRL-R expression in organs [bovine corpus luteum (CL), umbilical vein, aorta] and in organ-derived EC cultures. i Results: /i In the intact CL, both PRL-R forms were present at mRNA and protein level throughout the oestrous cycle stages. The short form prevailed as protein. PRL-R-positive EC were noted by immunofluorescent staining in arterial blood vessels of CL septa, in the umbilical vein and the aorta. In EC cultures of micro- and macrovascular origin, transcripts of both PRL-R forms were shown again the short-form protein prevailed. Blocking experiments with anti-prolactin (PRL) antibody led to a 60% decrease in cell growth. Treatment with PRL had no effect. i Conclusion: /i PRL-R expression in micro- and macrovascular EC is associated with the predominant short form.
Publisher: Springer Science and Business Media LLC
Date: 12-12-2011
DOI: 10.1007/S10585-010-9363-7
Abstract: The assembly of pericellular matrix containing hyaluronan (HA) and versican has been shown to be a pre-requisite for proliferation and migration of mesenchymal cells. In this study, we investigated whether treatment with recombinant versican could induce the formation of a pericellular matrix by ovarian cancer cells (OVCAR-3, OVCAR-5, and SKOV-3) and promote their motility, invasion, and adhesion to peritoneal cells in vitro. We also determined whether versican-induced pericellular matrix formation and metastatic cancer cell behavior could be blocked by small HA oligosaccharides. Only combined treatment with recombinant versican and HA resulted in pericellular matrix formation by OVCAR-5 and SKOV-3 but not by OVCAR-3 cells, which lack the HA receptor, CD44. The motility of OVCAR-5 and SKOV-3 cells was significantly increased in scratch wound and chemotaxis assays following treatment with recombinant versican and HA. Versican and HA also promoted invasion of SKOV-3 and OVCAR-5 cells but had no effect on OVCAR-3 cells. We have demonstrated that exogenous HA significantly increased OVCAR-5 and SKOV-3 adhesion to peritoneal cells but adhesion was not further increased by versican treatment. Small HA oligomers (6-10 disaccharides) were able to significantly block formation of pericellular matrix by OVCAR-5 cells, as well as the increased motility and invasion induced by recombinant versican. HA oligomers also significantly blocked OVCAR-5 adhesion to peritoneal cells both in the presence and absence of exogenous HA. The dependence of CD44 for the versican and HA mediated effects were demonstrated by the inhibition of pericellular matrix formation as well as motility and invasion of OVCAR-5 cells following treatment with CD44 neutralizing antibody in the presence of versican and HA. We conclude that the acquisition of a HA/versican pericellular matrix by ovarian cancer cells increases their metastatic potential. HA oligomers can block this mechanism and are promising inhibitors of ovarian cancer dissemination.
Publisher: Oxford University Press (OUP)
Date: 08-04-2015
DOI: 10.1017/S1431927615000380
Abstract: X-ray fluorescence (XRF) was used to image 40 histological cross-sections of bovine ovaries ( n =19), focusing on structures including: antral follicles at different stages of growth or atresia, corpora lutea at three stages of development (II–IV), and capillaries, arterioles, and other blood vessels. This method identified three key trace elements [iron (Fe), zinc (Zn), and selenium (Se)] within the ovarian tissue which appeared to be localized to specific structures. Owing to minimal preprocessing of the ovaries, important high-resolution information regarding the spatial distribution of these elements was obtained with elemental trends and colocalizations of Fe and Zn apparent, as well as the infrequent appearance of Se surrounding the antrum of large follicles, as previously reported. The ability to use synchrotron radiation to measure trace element distributions in bovine ovaries at such high resolution and over such large areas could have a significant impact on understanding the mechanisms of ovarian development. This research is intended to form a baseline study of healthy ovaries which can later be extended to disease states, thereby improving our current understanding of infertility and endocrine diseases involving the ovary.
Publisher: Springer Science and Business Media LLC
Date: 14-01-2014
Abstract: At later stages of folliculogenesis, the mammalian ovarian follicle contains layers of epithelial granulosa cells surrounding an antral cavity. During follicle development granulosa cells replicate, secrete hormones and support the growth of the oocyte. In cattle, the follicle needs to grow 10 mm in diameter to allow an oocyte to ovulate, following which the granulosa cells cease iding and differentiate into the specialised cells of the corpus luteum. To better understand the molecular basis of follicular growth and granulosa cell maturation, we undertook transcriptome profiling of granulosa cells from small ( 5 mm n = 10) and large ( 10 mm, n = 4) healthy bovine follicles using Affymetrix microarrays (24,128 probe sets). Principal component analysis for the first two components and hierarchical clustering showed clustering into two groups, small and large, with the former being more heterogeneous. Size-frequency distributions of the coefficient of variation of the signal intensities of each probe set also revealed that small follicles were more heterogeneous than the large. IPA and GO enrichment analyses revealed that processes of axonal guidance, immune signalling and cell rearrangement were most affected in large follicles. The most important networks were associated with: (A) Notch, SLIT/ROBO and PI3K signalling, and (B) ITGB5 and extracellular matrix signalling through extracellular signal related kinases (ERKs). Upstream regulator genes which were predicted to be active in large follicles included STAT and XBP1. By comparison, developmental processes such as those stimulated by KIT , IHH and MEST were most active in small follicles. MGEA5 was identified as an upstream regulator in small follicles. It encodes an enzyme that modifies the activity of many target proteins, including those involved in energy sensing, by removal of N-acetylglucosamine from serine and threonine residues. Our data suggest that as follicles enlarge more genes and/or pathways are activated than are inactivated, and gene expression becomes more uniform. These findings could be interpreted that either the cells in large follicles are more uniform in their gene expression, or that follicles are more uniform or a combination of both and that additional factors, such as LH, are additionally controlling the granulosa cells.
Publisher: Elsevier BV
Date: 03-2019
DOI: 10.1016/J.MEHY.2019.01.019
Abstract: Polycystic ovary syndrome (PCOS) affects around 10% of women of reproductive age and is most common in developed countries. The aetiology of PCOS is not completely understood. Current evidence suggests that the syndrome results from a genetic predisposition interacting with developmental events during fetal or perinatal life that together increase susceptibility in some in iduals. This implies that environmental factors influence the initiation of PCOS in the fetus or infant, either directly or via the mother. PCOS is often considered to be an ancient disorder but there is no direct proof of this in the medical or historic record. One of the cardinal features, polycystic ovaries, was first described only in the early 1900s, despite reports of many thousands of autopsies recorded earlier. This conundrum could be explained by postulating that polycystic ovaries were rare before the 1900s and have become more common over the last 100 years. The hypothesis that PCOS is a syndrome of the 20th Century would eliminate the need to explain the paradox of why there exists a genetic predisposition to subfertility syndrome.
Publisher: Bioscientifica
Date: 06-2019
DOI: 10.1530/REP-18-0323
Abstract: The ovary has specialised stromal compartments, including the tunica albuginea, interstitial stroma and theca interna, which develops concurrently with the follicular antrum. To characterise the molecular determinants of these compartments, stroma adjacent to preantral follicles (pre-theca), interstitium and tunica albuginea were laser microdissected ( n = 4 per group) and theca interna was dissected from bovine antral follicles ( n = 6). RNA microarray analysis showed minimal differences between interstitial stroma and pre-theca, and these were combined for some analyses and referred to as stroma. Genes significantly upregulated in theca interna compared to stroma included INSL3 , LHCGR , HSD3B1 , CYP17A1 , ALDH1A1 , OGN , POSTN and ASPN . Quantitative RT-PCR showed significantly greater expression of OGN and LGALS1 in interstitial stroma and theca interna versus tunica and greater expression of ACD in tunica compared to theca interna. PLN was significantly higher in interstitial stroma compared to tunica and theca. Ingenuity pathway, network and upstream regulator analyses were undertaken. Cell survival was also upregulated in theca interna. The tunica albuginea was associated with GPCR and cAMP signalling, suggesting tunica contractility. It was also associated with TGF-β signalling and increased fibrous matrix. Western immunoblotting was positive for OGN, LGALS1, ALDH1A1, ACD and PLN with PLN and OGN highly expressed in tunica and interstitial stroma (each n = 6), but not in theca interna from antral follicles ( n = 24). Immunohistochemistry localised LGALS1 and POSTN to extracellular matrix and PLN to smooth muscle cells. These results have identified novel differences between the ovarian stromal compartments.
Publisher: Informa UK Limited
Date: 10-2009
Abstract: Autophagic cell death has been observed in granulosa cell cultures via the oxLDL-dependent activation of lectin-like oxidized low density lipoprotein receptor 1 (LOX-1). This activation might differ for cytokeratin-positive (CK(+)) and CK(-) granulosa cells. In particular, LOX-1 and toll-like receptor 4 (TLR4), one of the pattern recognition receptors of innate immunity, might be ersely regulated. Granulosa cell subtype cultures were established from the follicle harvests of patients undergoing in vitro fertilization (IVF) therapy. In response to oxLDL treatment, the fibroblast-like CK(-) cells upregulated LOX-1 and exhibited reparative autophagy, which could be blocked with anti-LOX-1 antibody. The epithelioid-like CK(+) cells did not regulate LOX-1 expression upon oxLDL application, but the expression of TLR4 and CD14 increased between 0 and 36 h of oxLDL/nDL treatment. This upregulation was associated with nonapoptotic cell death based on the absence of cleaved caspase-3. Reactive oxygen species (ROS) increased with 12 h oxLDL application and steroidogenic acute regulatory (StAR) protein expression was negligible. In CK(-) cells, the inhibition of TLR4 downregulated LOX-1 and induced apoptosis. We concluded that CK(-) granulosa cells are protected against oxLDL-dependent apoptosis by TLR4, whereas, in CK(+) cells, oxLDL-induced TLR4 activation triggers nonapoptotic cell death. The CK(+) cells might represent immune-like granulosa cells involved in ovarian remodeling processes.
Publisher: Elsevier BV
Date: 2009
DOI: 10.1016/J.DIFF.2008.09.002
Abstract: We describe the use of rotary cultures (72 rpm) as an excellent method for generating spheroids from dispersed bovine granulosa cells (GC). The GC spheroids were symmetrical (diameter between 100 and 200 microm), easily accessible, and could be obtained at high yields. On day one, the spheroids showed a two-layered outer zone of cells that stained lighter than the inner zone in semi-thin sections. Bromodeoxyuridine (BrdU) uptake was frequent and randomly distributed. By day two, a striking decrease in BrdU uptake was noted. Apoptotic bodies appeared up to day four, as did TUNEL and propidium iodide labelled dead cells. At that time, the inner zone contained cells with large-sized vacuoles and the core was amorphous. The large-sized vacuoles were identified at the ultrastructural level and represented autophagosomes and autophagolysosomes that were in different stages of development. Surprisingly, conspicuous signs of cell death were accompanied by an increase in spontaneous luteinization compared to conventional stationary cultures. We detected high levels of progesterone (immunoassay) accompanied by high levels of the proteins and enzymes relevant for steroidogenesis (StAR, P450scc, 3beta-HSD by immunoblot and immunohistochemistry, respectively). Concomitant to cell death, GC spheroids augment progesterone synthesis. The GC spheroids provide an ideal model for studying steroidogenesis coupled to programmed cell death at the level of the mitochondria.
Publisher: American Chemical Society (ACS)
Date: 19-02-2020
Publisher: Public Library of Science (PLoS)
Date: 23-06-2014
Publisher: Springer Science and Business Media LLC
Date: 18-01-2014
Abstract: The major function of the ovary is to produce oocytes for fertilisation. Oocytes mature in follicles surrounded by nurturing granulosa cells and all are enclosed by a basal lamina. During growth, granulosa cells replicate and a large fluid-filled cavity (the antrum) develops in the centre. Only follicles that have enlarged to over 10 mm can ovulate in cows. In mammals, the number of primordial follicles far exceeds the numbers that ever ovulate and atresia or regression of follicles is a mechanism to regulate the number of oocytes ovulated and to contribute to the timing of ovulation. To better understand the molecular basis of follicular atresia, we undertook transcriptome profiling of granulosa cells from healthy (n = 10) and atretic (n = 5) bovine follicles at early antral stages ( 5 mm). Principal Component Analysis (PCA) and hierarchical classification of the signal intensity plots for the arrays showed primary clustering into two groups, healthy and atretic. These analyses and size-frequency plots of coefficients of variation of signal intensities revealed that the healthy follicles were more heterogeneous. Examining the differentially-expressed genes the most significantly affected functions in atretic follicles were cell death, organ development, tissue development and embryonic development. The overall processes influenced by transcription factor gene TP53 were predicted to be activated, whereas those of MYC were inhibited on the basis of known interactions with the genes in our dataset. The top ranked canonical pathway contained signalling molecules common to various inflammatory/fibrotic pathways such as the transforming growth factor-β and tumour necrosis factor-α pathways. The two most significant networks also reflect this pattern of tissue remodelling/fibrosis gene expression. These networks also contain molecules which are present in the canonical pathways of hepatic fibrosis/hepatic stellate cell activation and transforming growth factor-β signalling and were up regulated. Small healthy antral follicles, which have a number of growth outcomes, exhibit greater variability in gene expression, particularly in genes associated with cell ision and other growth-related functions. Atresia, on the other hand, not only involves cell death but clearly is an active process similar to wound healing.
Publisher: CSIRO Publishing
Date: 2020
DOI: 10.1071/RD19017
Abstract: Few studies have investigated the effects of nutrition during the periconception and early gestation periods on fetal and placental development in cattle. In this study, nulliparous yearling heifers (n=360) were in idually fed a diet high or low in protein (HPeri and LPeri) beginning 60 days before conception. From 24 to 98 days after conception, half of each treatment group was changed to the alternative high- or low-protein diet (HPost and LPost) yielding four groups in a 2×2 factorial design. A subset of heifers (n=46) was necropsied at 98 days after conception and fetoplacental development assessed. Placentome number and volume decreased in response to LPeri and LPost diets respectively. Absolute lung, pancreas, septum and ventricle weights decreased in LPost versus HPost fetuses, whereas the post-conception diet altered absolute and relative liver and brain weights depending on sex. Similarly, changes in fetal hepatic gene expression of factors regulating growth, glucose output and lipid metabolism were induced by protein restriction in a sex-specific manner. At term, neonatal calf and placental measures were not different. Protein restriction of heifers during the periconception and early gestation periods alters fetoplacental development and hepatic gene expression. These changes may contribute to functional consequences for progeny, but this may not be apparent from gross morphometry at birth.
Publisher: Public Library of Science (PLoS)
Date: 10-03-2017
Publisher: The Endocrine Society
Date: 02-2015
DOI: 10.1210/ER.2014-1079
Publisher: Public Library of Science (PLoS)
Date: 11-03-2019
Publisher: Oxford University Press (OUP)
Date: 2015
DOI: 10.1039/C5MT00035A
Abstract: Highlights how quantitative XRF can differentiate between biological structures in bovine ovaries on the basis of trace element distribution alone.
Publisher: Public Library of Science (PLoS)
Date: 22-03-2019
Publisher: SAGE Publications
Date: 19-12-2019
Abstract: When first formed, the ovary only has an established epithelium at its base or hilum. Later, an epithelium is established around the rest of the ovary. To examine this further, we conducted scanning electron microscopy of the surface of bovine fetal ovaries and immunohistochemistry of ovarian cross-sections. From the earliest time point, the cells on the surface of the base or hilum of the ovary were cuboidal. On the remainder of the ovary, the surface was more irregular. By mid-development, the surface was covered completely with either a stratified or simple epithelium of cuboidal cells. Clefts were observed in the surface and appeared to form due to the expansion of stroma surrounding each open ovigerous cord, elevating the areas surrounding each cord, while leaving the opening of the cord to form the base of each cleft. The continued expansion of the surrounding stroma below the surface appeared not only to close the ovigerous cords from the surface but to compress the clefts into the shape of a groove. Later, most of the ovarian surface was covered with a simple cuboidal epithelium. The changes to the ovarian surface during fetal development coincide with the remodeling of the stroma and cords below.
Publisher: Oxford University Press (OUP)
Date: 2015
DOI: 10.1039/C4MT00338A
Abstract: The first study of bromine speciation and distribution in mammalian tissues using X-rays shows bromine present predominantly as bromide.
Publisher: American Physiological Society
Date: 12-2017
DOI: 10.1152/AJPENDO.00280.2007
Abstract: In the corpus luteum (CL), blood vessels develop, stabilize, and regress. This process depends on the ratio of pro- and antiangiogenic factors, which change during the ovarian cycle. The present study focuses on the possible roles of 23,000 (23K) prolactin (PRL) in the bovine CL and its antiangiogenic NH 2 -terminal fragments after extracellular cleavage by cathepsin D (Cath D). PRL RNA and protein were demonstrated in the CL tissue, in luteal endothelial cells, and in steroidogenic cells. Cath D was detected in CL tissue, cell extracts, and corresponding cell supernatants. In the intact CL, 23K PRL levels decreased gradually, whereas Cath D levels concomitantly increased between early and late luteal stages. In vitro, PRL cleavage occurred in the presence of acidified homogenates of CL tissue, cells, and corresponding cell supernatants. Similar fragments were obtained with purified Cath D, and their appearance was inhibited by pepstatin A. The aspartic protease specific substrate MOCAc-GKPILF∼FRLK(Dnp)-d- R-NH 2 was cleaved by CL cell supernatants, providing further evidence for Cath D activity. The 16,000 PRL inhibited proliferation of luteal endothelial cells accompanied by an increase in cleaved caspase-3. In conclusion, 1) the bovine CL is able to produce PRL and to process it into antiangiogenic fragments by Cath D activity and 2) PRL cleavage might mediate angioregression during luteolysis.
Publisher: Wiley
Date: 16-03-2011
DOI: 10.1096/FJ.11-181099
Publisher: Bioscientifica
Date: 08-2016
DOI: 10.1530/REP-16-0172
Abstract: Fibrillins 1–3 are stromal extracellular matrix proteins that play important roles in regulating TGFβ activity, which stimulates fibroblasts to proliferate and synthesize collagen. In the developing ovary, the action of stroma is initially necessary for the formation of ovigerous cords and subsequently for the formation of follicles and the surface epithelium of the ovary. FBN3 is highly expressed only in early ovarian development and then it declines. In contrast, FBN1 and 2 are upregulated in later ovarian development. We examined the expression of FBN1–3 in bovine and human fetal ovaries. We used cell dispersion and monolayer culture, cell passaging and tissue culture. Cells were treated with growth factors, hormones or inhibitors to assess the regulation of expression of FBN1–3 . When bovine fetal ovarian tissue was cultured, FBN3 expression declined significantly. Treatment with TGFβ-1 increased FBN1 and FBN2 expression in bovine fibroblasts, but did not affect FBN3 expression. Additionally, in cultures of human fetal ovarian fibroblasts (9–17weeks gestational age), the expression of FBN1 and FBN2 increased with passage, whereas FBN3 dramatically decreased. Treatment with activin A and a TGFβ family signaling inhibitor, SB431542, differentially regulated the expression of a range of modulators of TGFβ signaling and of other growth factors in cultured human fetal ovarian fibroblasts suggesting that TGFβ signaling is differentially involved in the regulation of ovarian fibroblasts. Additionally, since the changes in FBN1–3 expression that occur in vitro are those that occur with increasing gestational age in vivo , we suggest that the fetal ovarian fibroblasts mature in vitro .
Publisher: Oxford University Press (OUP)
Date: 12-04-2022
Abstract: Could changes in transforming growth factor β (TGFβ) signalling during foetal ovary development alter the expression of polycystic ovary syndrome (PCOS) candidate genes leading to a predisposition to PCOS? TGFβ signalling molecules are dynamically expressed during foetal ovary development and TGFβ1 inhibits expression of the androgen receptor (AR) and 7 (INSR, C8H9orf3, RAD50, ERBB3, NEIL2, IRF1 and ZBTB16) of the 25 PCOS candidate genes in foetal ovarian fibroblasts in vitro, whilst increasing expression of the AR cofactor TGFβ-induced transcript 1 (TGFB1I1 or Hic5). The ovarian stroma arises from the mesonephros during foetal ovary development. Changes in the morphology of the ovarian stroma are cardinal features of PCOS. The ovary is more fibrous and has more tunica and cortical and subcortical stroma. It is not known why this is and when this arises. PCOS has a foetal origin and perhaps ovarian stroma development is altered during foetal life to determine the formation of a polycystic ovary later in life. PCOS also has a genetic origin with 19 loci containing 25 PCOS candidate genes. In many adult tissues, TGFβ is known to stimulate fibroblast replication and collagen deposition in stroma, though it has the opposite effect in the non-scaring foetal tissues. Our previous studies showed that TGFβ signalling molecules [TGFβs and their receptors, latent TGFβ binding proteins (LTBPs) and fibrillins, which are extracellular matrix proteins that bind LTBPs] are expressed in foetal ovaries. Also, we previously showed that TGFβ1 inhibited expression of AR and 3 PCOS candidate genes (INSR, C8H9orf3 and RAD50) and stimulated expression of TGFB1I1 in cultured foetal ovarian fibroblasts. We used Bos taurus for this study as we can ethically collect foetal ovaries from across the full 9-month gestational period. Foetal ovaries (62–276 days, n = 19) from across gestation were collected from pregnant B. taurus cows for RNA-sequencing (RNA-seq) analyses. Foetal ovaries from B. taurus cows were collected (160–198 days, n = 6) for culture of ovarian fibroblasts. RNA-seq transcriptome profiling was performed on foetal ovaries and the data on genes involved in TGFβ signalling were extracted. Cells were dispersed from foetal ovaries and fibroblasts cultured and treated with TGFβ1. The effects of TGFβ regulation on the remaining eight PCOS candidate genes not previously studied (ERBB3, MAPRE1, FDFT1, NEIL2, ARL14EP, PLGRKT, IRF1 and ZBTB16) were examined. Many TGFβ signalling molecules are expressed in the foetal ovary, and for most, their expression levels increased accross gestation (LTBP1/2/3/4, FBN1, TGFB2/3, TGFBR2/3 and TGFB1I1), while a few decreased (FBN3, TGFBR3L, TGFBI and TGFB1) and others remained relatively constant (TGFBRAP1, TGFBR1 and FBN2). TGFβ1 significantly decreased expression of PCOS candidate genes ERBB3, NEIL2, IRF1 and ZBTB16 in cultured foetal ovarian fibroblasts. The FASTQ files, normalized data and experimental information have been deposited in the Gene Expression Omnibus (GEO) accessible by accession number GSE178450. Regulation of PCOS candidate genes by TGFβ was carried out in vitro and further studies in vivo are required. This study was carried out in bovine where foetal ovaries from across all of the 9-month gestational period were available, unlike in the human where it is not ethically possible to obtain ovaries from the second half of gestation. From our current and previous results we speculate that inhibition of TGFβ signalling in the foetal ovary is likely to (i) increase androgen sensitivity by enhancing expression of AR, (ii) increase stromal activity by stimulating expression of COL1A1 and COL3A1 and (iii) increase the expression of 7 of the 25 PCOS candidate genes. Thus inhibition of TGFβ signalling could be part of the aetiology of PCOS or at least the aetiology of polycystic ovaries. Funding was received from Adelaide University China Fee Scholarship (M.L.), Australian Research Training Program (R.A.) and the Faculty of Health and Medical Science Divisional Scholarship (R.A.), Adelaide Graduate Research Scholarships (R.A. and N.A.B.), Australia Awards Scholarship (M.D.H.), Robinson Research Institute Career Development Fellowship (K.H.) and Building On Ideas Grant (K.H.), National Health and Medical Research Council of Australia Centre for Research Excellence in the Evaluation, Management and Health Care Needs of Polycystic Ovary Syndrome (N.A.B., M.D.H. and R.J.R. GTN1078444) and the Centre for Research Excellence on Women’s Health in Reproductive life (R.A., R.J.R. and K.H. GTN1171592) and the UK Medical Research Council (R.A.A. grant no. G1100357). The funders did not play any role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. The authors of this manuscript have nothing to declare and no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Publisher: Public Library of Science (PLoS)
Date: 15-05-2014
Publisher: Oxford University Press (OUP)
Date: 2015
DOI: 10.1039/C4MT90049A
Abstract: Correction for 'X-Ray fluorescence imaging and other analyses identify selenium and GPX1 as important in female reproductive function' by M. J. Ceko et al., Metallomics, 2014, DOI: .
Publisher: Elsevier BV
Date: 11-2012
DOI: 10.1016/J.MCE.2012.07.009
Abstract: In the ovarian follicular membrana granulosa there are morphological and functional differences between cells adjacent to the follicular fluid lumen, or aligning the basal lamina. Amongst the observed functional differences are steroidogenic capacity and expression levels of a novel basal lamina, focimatrix both of which increase in the later stages of antral follicle growth. A number of different studies have produced apparently inconsistent results as to which cell layers are more steroidogenic. To examine this systematically, in idual bovine follicles, confirmed as healthy by post hoc histological examination, were used to isolate populations of apical and basal granulosa cells. Cell counts revealed that the respective groups did not differ in the numbers of cells, thus confirming the separation of these populations. We measured gene expression (quantitative RT-PCR, n=8-10, follicle diameter 14.0±0.5 mm) and protein levels (Western immunoblotting, n=14, follicle diameter 11.9±0.5 mm) and hormone production from granulosa cells (2.5×10(5) viable cells/well in serum-free conditions for 24 h, n=15, diameter 12±0.5 mm). Levels of mRNA of HSD3B1 and CYP19A1 and three focimatrix genes COL4A1, HSPG2 and LAMB2 and LHCGR were significantly lower in apical granulosa cells (P 0.05). The protein levels of steroidogenic enzymes P450scc and P450arom were significantly higher in apical cells (P 0.05). Progesterone production was significantly lower and oestradiol production was significantly higher in apical granulosa cells (P<0.05). These results confirm that apical and basal cells are functionally different, and the differences might be explained by the location of cells of different ages and maturity within the membrana granulosa. Discrepancies in the literature on their steroidogenic capacity may reflect differences in the steroidogenic parameters measured.
Publisher: Elsevier BV
Date: 2012
Publisher: Elsevier BV
Date: 05-2008
DOI: 10.1016/J.EJCB.2008.01.005
Abstract: The protease cathepsin D (Cath D) and its proteolytically inactive proform, procathepsin D (ProCath D), turned out to be multifunctional within and outside the cell. Elevated levels of ProCath D occur in malignant tumors and in organs under chronic inflammation. One important source for this increase of ProCath D might be endothelial cells. Here we examined the expression of Cath D in the human endothelial cell line EA.hy 926 and in primary endothelial cells isolated from human umbilical cord veins (HUVEC). After serum-free incubation with or without human interferon-gamma (hIFN-gamma) and/or human tumor necrosis factor-alpha (hTNF-alpha) immature and mature Cath D forms were examined in cell extracts and in cell-conditioned medium concentrates by Western blotting. Lysates of EA.hy 926 cells as well as of HUVEC contained active Cath D as two-chain form, but only negligible amounts of ProCath D and Cath D intermediates. Yet both endothelial cell cultures accumulated ProCath D in their conditioned media in the absence of any stimulus. The treatment with hIFN-gamma and/or hTNF-alpha had little effect on intracellular levels of Cath D, whereas the cytokine stimulation increased the extracellular presence of ProCath D in both endothelial cell cultures. The extracellular increase of ProCath D was not related to induction of apoptosis, as validated by cleaved caspase-3 in cell lysates. Acidification of cytokine-treated media converted ProCath D into Cath D, which was associated with cathepsin-like activity using a fluorogenic substrate-linked assay. We conclude, in vitro, endothelial cells are a cytokine-dependent source for extracellular ProCath D.
Publisher: SAGE Publications
Date: 2014
Abstract: The isolation of islets by collagenase digestion can cause damage and impact the efficiency of islet engraftment and function. In this study, we assessed the basement membranes (BMs) of mouse pancreatic islets as a molecular biomarker for islet integrity, damage after isolation, and islet repair in vitro as well as in the absence or presence of an immune response after transplantation. Immunofluorescence staining of BM matrix proteins and the endothelial cell marker platelet endothelial cell adhesion molecule-1 (PECAM-1) was performed on pancreatic islets in situ, isolated islets, islets cultured for 4 days, and islet grafts at 3–10 days posttransplantation. Flow cytometry was used to investigate the expression of BM matrix proteins in isolated islet β-cells. The islet BM, consisting of collagen type IV and components of Engelbreth–Holm–Swarm (EHS) tumor laminin 111, laminin α2, nidogen-2, and perlecan in pancreatic islets in situ, was completely lost during islet isolation. It was not reestablished during culture for 4 days. Peri- and intraislet BM restoration was identified after islet isotransplantation and coincided with the migration pattern of PECAM-1 + vascular endothelial cells (VECs). After islet allotransplantation, the restoration of VEC-derived peri-islet BMs was initiated but did not lead to the formation of the intraislet vasculature. Instead, an abnormally enlarged peri-islet vasculature developed, coinciding with islet allograft rejection. The islet BM is a sensitive biomarker of islet damage resulting from enzymatic isolation and of islet repair after transplantation. After transplantation, remodeling of both peri- and intraislet BMs restores β-cell–matrix attachment, a recognized requirement for β-cell survival, for isografts but not for allografts. Preventing isolation-induced islet BM damage would be expected to preserve the intrinsic barrier function of islet BMs, thereby influencing both the effector mechanisms required for allograft rejection and the antirejection strategies needed for allograft survival.
Publisher: CSIRO Publishing
Date: 2018
DOI: 10.1071/RD18218
Abstract: During ovarian development stroma from the mesonephros penetrates and expands into the ovarian primordium and thus appears to be involved, at least physically, in the formation of ovigerous cords, follicles and surface epithelium. Cortical stromal development during gestation in bovine fetal ovaries (n=27) was characterised by immunohistochemistry and by mRNA analyses. Stroma was identified by immunostaining of stromal matrix collagen type I and proliferating cells were identified by Ki67 expression. The cortical and medullar volume expanded across gestation, with the rate of cortical expansion slowing over time. During gestation, the proportion of stroma in the cortex and total volume in the cortex significantly increased (P& .05). The proliferation index and numerical density of proliferating cells in the stroma significantly decreased (P& .05), whereas the numerical density of cells in the stroma did not change (P& .05). The expression levels of 12 genes out of 18 examined, including osteoglycin (OGN) and lumican (LUM), were significantly increased later in development (P& .05) and the expression of many genes was positively correlated with other genes and with gestational age. Thus, the rate of cortical stromal expansion peaked in early gestation due to cell proliferation, whilst late in development expression of extracellular matrix genes increased.
Publisher: Springer Science and Business Media LLC
Date: 28-01-2014
Abstract: Oocytes mature in ovarian follicles surrounded by granulosa cells. During follicle growth, granulosa cells replicate and secrete hormones, particularly steroids close to ovulation. However, most follicles cease growing and undergo atresia or regression instead of ovulating. To investigate the effects of stimulatory (follicle-stimulating hormone FSH) and inhibitory (tumour necrosis factor alpha TNFα) factors on the granulosa cell transcriptome, bovine ovaries were obtained from a local abattoir and pools of granulosa cells were cultured in vitro for six days under defined serum-free conditions with treatments present on days 3–6. Initially dose–response experiments (n = 4) were performed to determine the optimal concentrations of FSH (0.33 ng/ml) and TNFα (10 ng/ml) to be used for the microarray experiments. For array experiments cells were cultured under control conditions, with FSH, with TNFα, or with FSH plus TNFα (n = 4 per group) and RNA was harvested for microarray analyses. Statistical analysis showed primary clustering of the arrays into two groups, control/FSH and TNFα/TNFα plus FSH. The effect of TNFα on gene expression dominated that of FSH, with substantially more genes differentially regulated, and the pathways and genes regulated by TNFα being similar to those of FSH plus TNFα treatment. TNFα treatment reduced the endocrine activity of granulosa cells with reductions in expression of FST , INHA , INBA and AMH. The top-ranked canonical pathways and GO biological terms for the TNFα treatments included antigen presentation, inflammatory response and other pathways indicative of innate immune function and fibrosis. The two most significant networks also reflect this, containing molecules which are present in the canonical pathways of hepatic fibrosis/hepatic stellate cell activation and transforming growth factor β signalling, and these were up regulated. Upstream regulator analyses also predicted TNF, interferons γ and β1 and interleukin 1β. In vitro , the transcriptome of granulosa cells responded minimally to FSH compared with the response to TNFα. The response to TNFα indicated an active process akin to tissue remodelling as would occur upon atresia. Additionally there was reduction in endocrine function and induction of an inflammatory response to TNFα that displays features similar to immune cells.
Publisher: Informa UK Limited
Date: 2009
DOI: 10.1080/08977190802707571
Abstract: We report the presence of KIT variants in granulosa and thecal cells of the follicle and endothelial and steroidogenic cells of the corpus luteum. Transcripts of both full-length splice variants, KIT and KITA, were ubiquitously detected in all cell types, in contrast to transcripts for truncated KIT. RT-PCR with exon-intron-specific primers suggested that KIT transcripts retained intron sequences. We used domain-specific KIT antibodies to identify truncated KIT proteins in cell conditioned media and lysates. These proteins represented soluble KIT and a so far disregarded intracellular KIT fragment, and were ubiquitously present. In contrast, glycosylated variants of full-length KIT were predominantly detected in thecal and endothelial cells. All KIT variants were encountered again in COS-7 cells transfected with a vector containing KITA. Phorbol 12-myristate-13-acetate treatment induced levels of truncated KITs, and this effect was repressed by the metalloproteinase inhibitor TAPI-1. Our findings show that ectodomain cleavage of full-length KIT generates an intracellular KIT. Our experiments suggest that replenishing full-length KIT differs among various ovarian cell types.
Publisher: Oxford University Press (OUP)
Date: 2015
DOI: 10.1039/C4MT00228H
Abstract: Using XRF imaging as a path-finding experiment, we mapped the distribution of trace elements in sections of bovine ovaries the first study of its kind in mammalian ovaries.
Publisher: Springer Science and Business Media LLC
Date: 23-12-2010
Publisher: MDPI AG
Date: 11-08-2020
DOI: 10.3390/MOLECULES25163661
Abstract: Thiones have been investigated as ligands in metal complexes with catalytic and biological activity. We report the synthesis, characterization, and biological evaluation of a series of MII/III complexes of the general formulae [MII(cym)(L)Cl]X (cym = η6-p-cymene) or [MIII(Cp*)(L)Cl]X (Cp* = η5-pentamethylcyclopentadienyl), where X = Cl− or PF6−, and L represents heterocyclic derivatives of thiourea. The thiones feature a benzyl-triazolyl pendant and they act as bidentate ligands via N,S-coordination to the metal centers. Several derivatives have been investigated by single-crystal X-ray diffraction analysis. NMR investigations showed a counterion-dependent shift of several protons due to the interaction with the counterions. These NMR investigations were complemented with X-ray diffraction analysis data and the effects of different counterions on the secondary coordination sphere were also investigated by DFT calculations. In biological studies, the Ir benzimidazole derivative was found to accumulate in the cytoplasm and it was the most cytotoxic derivative investigated.
No related grants have been discovered for Katja Hummitzsch.